The activity of cells is regulated by external signals that stimulate or inhibit intracellular events. The process by which stimulatory or inhibitory signals are transmitted into and within a cell to elicit an intracellular response is referred to as signal transduction. Proper signal transduction is essential for proper cellular function. Defects in various components of signal transduction pathways, from cell surface receptors to activators of gene transcription, account for a vast number of diseases, including numerous forms of cancer, vascular diseases and neuronal diseases.
Protein kinases are enzymes involved in signal transduction which phosphorylate other proteins and/or themselves (autophosphorylation). Protein kinases involved in signal transduction in eukaryotic cells can be divided into three major groups based upon their substrate utilization: protein-tyrosine specific kinases (which phosphorylate substrates on tyrosine residues), protein-serine/threonine specific kinases (which phosphorylate substrates on serine and/or threonine residues) and dual-specificity kinases (which phosphorylate substrates on tyrosine, serine and/or threonine residues). Well over a hundred protein kinases have been identified to date and more are being identified at a very fast rate (about 10 to 30 new kinases per year) through genetic and molecular biological approaches. There are conserved regions among all protein kinases, suggesting evolution from a common ancestor. For reviews on protein kinases see Kemp, B. E. (ed.), (1990) Peptides and Protein Phosphorylation. CRC Press Inc. and Hanks et al. (1988) Science 241:42-52.
In order to insure fidelity in intracellular signal transduction cascades it is essential that each protein kinase have exquisite specificity in downstream targets. In some cases a kinase may have only a single substrate in a cell, but in general kinases appear to have a collection of targets that allow branching of an initial signal delivered to a cell in multiple directions in order to coordinate a set of events that occur in parallel for a given cellular response (see Roach, P. J. (1991) J. Biol. Chem. 266:14139-14142). The substrate specificity of a protein kinase can be influenced by at least three general mechanisms that depend on the overall structure of the enzyme: 1) specific domains in certain protein kinases target them to specific locations in the cell, thereby restricting their substrate availability; 2) other domains in the kinase, distinct from the catalytic domain, may provide high affinity association with either the substrate or an adapter molecule that presents the substrate to the kinase; and 3) specificity is ultimately provided by the structure of the catalytic site of the protein kinase.
Although the number of protein kinases that have been implicated in intracellular signaling is quite large, in only a few cases is there information about the sequence specificity of these kinases. This information has usually come from locating the phosphorylation sites on in vivo and/or in vitro substrates of the kinase. (For examples see Taylor et al., (1990) Ann. Rev. Biochem. 59:971-1005; Cheng, et al. (1991) J. Biol. Chem. 266:17919-17925; Walsh et al. (1990) in Peptides and Protein Phosphorylation. (B. E. Kemp, ed.) CRC Press Inc.pp. 43-84; Gaehlen and Harrison (1990) in Peptides and Protein Phosphorylation. (B. E. Kemp, ed.) CRC Press Inc.pp. 239-254). By comparing the sequences of several phosphorylation sites for a kinase a consensus sequence for substrates of that kinase can be determined. These types of studies have demonstrated the importance of the primary amino acid sequence around the site of phosphorylation in determining the in vivo specificity of protein kinases. Synthetic peptides can be constructed based upon the consensus sequence motif of a known phosphorylation site and individual amino acids can then be replaced one by one in order to determine the importance of particular amino acids on the K.sub.M or V.sub.max of the phosphorylation reaction.
However, there are severe limitations to this approach for determining the substrate specificity of a protein kinase. The procedure is quite expensive and laborious since each amino acid residue within a phosphorylation site must be altered individually to evaluate its importance. This approach does not necessarily identify all the residues critical for substrate specificity and, furthermore, an optimal substrate sequence is not likely to be determined unless each residue is changed to every other possible amino acid residue individually and then evaluated. For example, based upon an estimation of 9 to 12 amino acid residues of a substrate contacting the active site cleft of a kinase there would be approximately 1.024.times.10.sup.13 (20.sup.10) distinct peptides to consider. Moreover, in many cases this approach is not feasible because in vivo substrates for some kinase cannot be determined with certainty. For example, if an extracellular signal activates multiple kinases which phosphorylate multiple substrates or if a signal activates a cascade of kinases it is difficult to determine which kinase phosphorylates which substrate. An even more difficult problem in determining the substrate specificity of certain kinases is that their critical in vivo substrates are often proteins which are present in vivo in very low abundance and thus are not easily detectable by in vivo phosphorylation assays. Even relatively abundant substrates can be overlooked because of their high rates of dephosphorylation by closely-associated phosphatases.
Thus, there is a need for an alternative method to that of isolating and examining the sequences of native substrates for determining the substrate specificity of a protein kinase. Consensus sequence motifs for the phosphorylation sites of many known protein kinases have not yet been determined because of the limitations to current approaches discussed above. Information on the substrate specificity of each protein kinase involved in signal transduction would provide insight into signal transduction mechanisms and could allow for the design of novel therapeutic agents based on the substrate specificities of different protein kinases.